Ribosomal perturbation as a mechanism to prevent misfolding of CFTR

摘要 囊性纤维化（CF）是一种常见的致死性常染色体隐性遗传疾病，在美国每2，500至3，500人中发生1例。 每年出生。这种疾病是由CF跨膜传导调节因子（CFTR）缺陷引起的。 改变CFTR编码序列的突变改变了肽折叠的完整性并导致疾病。在这 项目，我们表明，CFTR密码子的改变，包括非同义和同义 常见的F508del变异体背景上的多态性可以扰乱核糖体动力学， mRNA利用率、翻译速率和蛋白质生物合成。蛋白质折叠与蛋白质结构之间的关键关系 和翻译速度是一个局部领域的分支，从基本的CFTR运输到疾病 表型和干预。这种应用的机制基础来自全基因组 使用酿酒酵母缺失菌株库进行表型筛选以鉴定新型的调节剂 F508del CFTR成熟，导致发现影响F508del的特定核糖体蛋白模块 CFTR折叠。我们已经确定Rpl 12的抑制，沿着其他60 S蛋白，其包括： 核糖体柄，提高F508del CFTR加工和活性在哺乳动物细胞表面的水平（在 原发性气道上皮细胞），预计可在CF患者中提供临床获益，且程度相当 最近批准用于这一目的的一种新药剂卢麦卡弗特。我们的初步数据显示， F508delCFTR的折叠和活性归因于对翻译动力学的影响。我们提出了三 具体目标是开发与CF发病机制相关的创新模型：1）定义核糖体结构域， 功能途径的相互作用，支配BMPF蛋白的生物发生，并扩大分析，包括其他 CFTR变体和复杂等位基因，2）确定Rpl12抑制拯救F508del的机制 CFTR功能，包括翻译率与导致疾病的CF突变和其他CFTR的相关性 3）通过RPL 12条件性敲除的发展确定体内意义，或 haplosufficient小鼠我们的团队结合了CFTR的多学科和相互加强的专业知识 生物化学，运输生理学，核糖体分析，翻译速度，酵母表型组学和分子生物学 遗传学，以机械地解决有关核糖体利用mRNA的方式， 影响所得基因产物的折叠和功能质量。我们将建立翻译控制， Yor1和CFTR成熟的新的和关键的介质，确定影响细胞增殖的特定核糖体模块。 CFTR生物合成途径，并评估在鼠疾病中抑制该靶点的相关性和安全性 模型这些结果将有助于加深对CF分子机制的理解，为CF的分子生物学研究提供新的途径。 个性化治疗CF患者最常见的疾病形式，并指出临床 一项与异常蛋白质翻译和一种致命遗传疾病有关的重要新观察结果的意义。

Abstract Cystic Fibrosis (CF) is a common lethal autosomal recessive disorder, occurring in 1 per 2,500 to 3,500 U.S. births annually. The disease is caused by defects in the CF transmembrane conductance regulator (CFTR). Mutations that change the CFTR coding sequence alter integrity of peptide folding and lead to disease. In this project, we show that CFTR codon alterations—including both non-synonymous and synonymous polymorphisms on background of the common F508del variant—can perturb ribosome dynamics, consequent mRNA utilization, translational rate, and protein biogenesis. The critical relationship between protein folding and translational velocity is a topical area with ramifications ranging from basic CFTR trafficking to disease phenotype and intervention. Mechanistic underpinnings for this application are derived from genome-wide phenomic screening with the Saccharomyces cerevisiae deletion strain library to identify novel modulators of F508del CFTR maturation, leading to discovery of specific ribosomal protein modules that impact F508del CFTR folding. We have established that suppression of Rpl12, along with other 60S proteins comprising the ribosomal stalk, improve F508del CFTR processing and activity at the mammalian cell surface to levels (in primary airway epithelia) predicted to confer clinical benefit among CF patients and comparable in magnitude to lumacaftor, a new agent recently approved for this purpose. Our preliminary data indicate that improved folding and activity of F508del CFTR are attributable to effects on translational kinetics. We propose three Specific Aims to develop an innovative model relevant to CF pathogenesis: 1) Define ribosomal domains and functional pathway interactions that govern ∆F protein biogenesis, and expand the analysis to include other CFTR variants and complex alleles, 2) Ascertain the mechanism by which Rpl12 suppression rescues F508del CFTR function, including relevance of translation rate to disease causing CF mutations and other CFTR polymorphisms, 3) Determine in vivo significance by development of RPL12 conditional knockout or haplosufficient mice. Our team combines multidisciplinary and mutually reinforcing expertise in CFTR biochemistry, transport physiology, ribosome profiling, translational velocity, yeast phenomics, and molecular genetics to mechanistically address a fundamental hypothesis regarding ways the ribosome utilizes mRNA to influence folding and functional quality of resulting gene products. We will establish translation control as a novel and critical mediator of Yor1 and CFTR maturation, identify specific ribosome modules that influence the CFTR biogenesis pathway, and evaluate relevance and safety of repressing this target in a murine disease model. Such results will improve understanding of CF molecular mechanism, suggest novel approaches for personalized treatment of CF patients with the most common forms of the disease, and indicate clinical significance of an important new observation relevant to aberrant protein translation and a fatal genetic illness.